The acquired enamel pellicle is a protein film readily formed on tooth mineral surfaces in the oral environment. It has been established that its formation is driven by the adsorption of proteins and peptides derived from oral fluid. Most of these proteins originate from either parotid or submandibular/ sublingual secretions which undergo modifications in the oral cavity before or after adsorption to the hydroxyapatite crystallites of tooth enamel. The composition and the structure of this acquired enamel pellicle are still largely unknown but play functionally a vital role with important physiological/clinical implications. A major thrust of this application is to use state-of-the-art proteomics to identify and characterize the major components of the in-vivo formed pellicle. Since the pellicle has an inner aspect facing the tooth surface and an outer aspect connected to the bacterial biofilm known as Dental plaque the functions of pellicle are multifaceted and complex. Another major goal is to investigate the protective functions of the pellicle vis-a-vis the maintenance of the mineral phase of enamel and the interplay between molecular entities of the pellicle and those bacteria which constitute the early colonizers of the biofilm forming on the tooth surface. The nature of the pellicle with respect to the early attachment of bacteria including those which are known periodontal pathogens will be studied in vitro and in vivo. The Specific Aims of the project are to: 1) Characterize components from pellicles formed in vivo by a variety of isolation techniques including 2D-electrophoresis followed by proteomic analyses comprising MALDI-TOF MS. LC-ESI MS and LC-MS/MS; 2) Explore the protective functions of the enamel pellicle by determining the affinity of its components to hydroxyapatite, the inhibitory potential of pellicle proteins/peptides of calcium phosphate precipitation from supersaturated solutions, and the capacity to retard demineralization; 3) Determine in vitro binding parameters of individual early pellicle colonizers, assess the role of transglutaminase in pellicle-bacterial interactions and employ biomimetic approaches to modify functional aspects of pellicle components; 4) Investigate with the checkerboard DNA-DNA hybridization assay the in vivo binding of early pellicle colonizers in healthy individuals and gingivitis patients, assess the relative role of exocrine and serum derived proteins/peptides on pellicle colonization in vivo and explore effects of different local environments in the oral cavity on these processes.